The goal of the competitive renewal is to test the hypothesis that in the hypoxic niche of the intervertebral disc, notch signaling is required for maintenance of proliferation and differentiation of cell populations. The objective of the first Specific Aim is to characterize the expression of notch and notch ligands and to investigate the importance of notch signaling in proliferation and differentiation of disc cells; to assess notch and notch ligand expression in tissue isolated from patients with degenerative disc disease. Using loss and gain of function studies we will determine the importance of the notch system in terms of cell proliferation and acquisition of the mature phenotype. By evaluating the localization of notch receptors and ligands we will gain insight into the interaction between different cell types within the disc. The goal of the second Specific Aim is to ascertain if in the hypoxic microenvironment notch and HIF signaling co-regulate expression of selective notch receptors, ligands and notch target genes in disc cells; in addition, we will determine if notch target genes Hey1 and Hey2 influence HIF function. In parallel, the interaction between notch and HIF will be assessed to evaluate the involvement of notch-ICD in the regulation of HIF-1 target genes. A final goal is to examine proliferative cells isolated from degenerate human discs for the expression and activities of notch signaling components. In the third Aim we will use a notochord specific knockout mice to investigate the importance of notch signaling in governing cell proliferation, survival and function during disc development and to delineate the role of notch in modulating HIF-1 function in the disc. Since we will also delete HIF-1, these studies will reveal the importance of this gene in regulating notch activity. The goal of the last Specific Aim is to develop an animal model to learn if inactivation of notch activity promotes a loss of cellularity, a decrease in number of differentiated cells and eventual degeneration of the intervertebral disc. We will inject rabbit intervertebral discs with lentivirus encoding psen1/psen2-SiRNA to suppress expression of endogenous notch activity. We will measure the activities of components of the notch signaling system, HIF function, the activities and numbers of proliferative cells and assess the degenerative status of the disc itself. These studies will validate and further extend the investigations described above, and provide new information on the critical role of notch signaling in terms of proliferation, survival, and differentiation of disc cells. Results of the investigation should provide a new understanding of how cell proliferation is regulated in the disc and provide a strategy for future use of pharmacological agents, and molecular genetic or tissue engineering approaches for effecting tissue repair utilizing endogenous disc cells.